Date of Award

7-1-1996

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biochemistry and Molecular Biology

Department

Graduate School of Biomedical Sciences

First Advisor

Stephen R. Grant

Abstract

Rege, Vaishali D., Mechanistic Studies of O-Acetylserine Sulfhydrylase-A from Salmonella typhimurium by Site-Directed Mutagenesis. Doctor of Philospophy (Biochemistry), July 1996, 94 pp., 3 tables, 27 illustrations, bibliography, 64 titles. O-Acetlyserine sulfhydrylase (OASS) is a pyridoxal 5’-phosphate dependent enzyme that catalyzes a β-replacement reaction forming L-cysteine and acetate from O-acetyl-L-serine (OAS) and sulfide. The pyridoxal 5’-phosphate (PLP) is bound at the active site in Schiff base linkage with a lysine. In the present study, the Schiff base lysine was identified as lysine 42 and its role in the OASS reaction was determined by changing it to alanine using site directed mutagenesis. K42A-OASS is isolated as an external aldimine with methionine or leucine and shows no reaction with the natural substrates. Apo-K42A-OASS can be reconstituted with PLP suggesting that K42 is not necessary for cofactor binding and formation of the external Schiff base. The apo-K42A-OASS, reconstituted with PLP, shows slow formation of the external aldimine but does not form the α-aminoacrylate intermediate on addition of OAS suggesting that K42 is involved in the abstraction of the α-proton in the β-elimination reaction. The external aldimine formed upon addition L-ala or L-ser are stable and represent a tautomer that absorbs maximally at 20 nm, while L-cys gives a tautomeric form of the external aldimine that absorbs at 330 nm, also seen in the overall reaction after addition of primary amines to the assay system. The use of a small primary amine such as ethylamine in the assay system. The use of a small primary amine such as ethylamine in the assay system or aminoethylation of C43 in apo-K42A-OASS reconstituted with PLP leads to the initial formation of an internal aldimine followed by the slow formation of the α-aminoacrylate intermediate on addition of OAS. Activity could not be fully recovered suggesting a significant rate enhancement from the presence of K42 for transamination and general base catalysis. Cysteine 43 is the only cysteine in the enzyme OASS-A, and that is next to the internal Schiff base lysine (K42). C43 has been replaced with alanine (C43A), serine (C43S) or threonine (C43T) by site-directed mutagenesis. Also, tryptophan 51 has been replaced with phenylalanine (W51F) or tyrosine (W51Y) by site-directed mutagenesis. Tryptophan 51 is one of the two tryptophan residues in the enzyme, and was thought to be responsible for fluorescence energy transfer to the PLP-internal Schiff base. The growth patterns of the strains carrying mutantions were compared with that of strain DW378 of S. typhimurium carrying plasmid pRSM40 with the cysK gene encoding OASS-A with no mutation. The growth patterns of the strains with mutations of C43A and C43S were very similar to that of pRSM40 whereas the strains with mutations at C43A and C43S were very similar to that of pRSM40 whereas the strains carrying mutations at W51F, W51Y and C43T showed very slow growth. The purification procedures for all of the mutant enzymes were similar to that of the wild type enzyme.

Comments

Rege, Vaishali D., Mechanistic Studies of O-Acetylserine Sulfhydrylase-A from Salmonella typhimurium by Site-Directed Mutagenesis. Doctor of Philospophy (Biochemistry), July 1996, 94 pp., 3 tables, 27 illustrations, bibliography, 64 titles.

Share

COinS